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. 2022 Feb 14;23(4):e53639. doi: 10.15252/embr.202153639

Figure 4. SCAI interacts with Polζ but protects against erroneous ICL repair independently of Polζ.

Figure 4

  1. Analysis of SCAI‐interacting proteins in Xenopus egg extracts. Volcano plot shows enrichment of proteins in both SCAI‐C and SCAI‐N immunoprecipitates (IPs) compared to an IgG control, plotted against the P‐value (n = 4 independent experiments; permutation‐based FDR < 0.01, s0  = 2).
  2. SCAI, REV7, REV1, and REV3 immunoprecipitates (IP) in NPE were immunoblotted with the indicated antibodies. Asterisks denote antibody cross‐reactivity.
  3. Whole proteome MS analysis of mock‐ versus SCAI‐depleted egg extracts. The volcano plot shows the difference in abundance of proteins between the mock reaction and SCAI‐depleted samples (with either N or C antibodies) (X‐axis), plotted against the P‐value resulting from two‐tailed Student’s t‐test (Y‐axis). Proteins significantly down‐regulated (FDR < 5%) in SCAI‐depleted extracts are highlighted in red (n = 4 biochemical replicates; FDR < 5% corresponds to a permutation‐based FDR‐adjusted q‐value of < 0.05).
  4. GFP IPs from U2OS or U2OS/GFP‐SCAI cells transfected with indicated siRNAs were immunoblotted with indicated antibodies. Note that a small fraction of REV7 is still immunoprecipitated with GFP‐SCAI in REV3 knockdown cells. This could be due to incomplete knockdown, and/or additional interactions of SCAI with the REV1‐Polζ complex that are independent of REV3.
  5. Schematic of SCAI interaction with the REV1‐Polζ complex. The SCAI‐interacting region of human REV3 and its alignment with a homologous region in human AHDC is indicated. NTD, N‐terminal domain; PCD, positively charged domain; Pol, polymerase domain; CTD, C‐terminal domain.
  6. GFP IPs from U2OS cells transfected with indicated REV3‐GFP expression constructs were immunoblotted with indicated antibodies.
  7. As in (F), except that U2OS or U2OS/GFP‐SCAI cells were transfected with FLAG‐NLS‐REV3(995–1194) expression plasmid.
  8. Mock‐, SCAI‐, REV1‐, or SCAI/REV1‐depleted egg extracts were analyzed by immunoblotting with indicated antibodies. Asterisks denote antibody cross‐reactivity.
  9. pICLpt was replicated in egg extracts immunodepleted with the indicated antibodies and processed as in Fig 3J and L.
  10. pICLpt was replicated in egg extracts immunodepleted with indicated antibodies, isolated at various times via plasmid pull down, and associated proteins were analyzed by immunoblotting.